Microbial transformation process for the preparation of hydroxylanol imidazo (4,5-b) pyridines useful as angiotensin II receptor antagonists

ABSTRACT

Fermentation of the microoganism Actinoplanacete sp. in the presence of the angiotensin II receptor antagonist, 5,7-dimethyl-2-ethyl-3-((2&#39;-tetrazol-5-yl)biphenyl-4-yl)methyl-3H-imidazo[4,5-b]pyridine, compound III, ##STR1## yields 2-ethyl-5-hydroxymethyl-7-methyl-3-((2&#39;-tetrazol-5-yl)biphenyl-4-yl)methyl-3H-imidazo[4,5-b]pyridine, compound I, and 2-ethyl-7-hydroxymethyl-5-methyl-3-(2&#39;-tetrazol-3-((2&#39;-tetrazol-5-yl)biphenyl-4-yl)methy-3H-imidazo[4,5-b]pyridine, compound II, ##STR2## both of which are angiotensin II receptor antagonists useful in the treatment of hypertension and congestive heart failure and other indications known to respond to angiotensin II antagonists.

RELATED APPLICATION

The present application is a divisional application of Ser. No. 539,941filed Jun. 18, 1990now abandoned, which is a continuation-in-partapplication of copending application Ser. No. 07/521,970, filed May 11,1990, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a novel process for the preparation of twoantihypertensive agents, compounds (I) and (II) ##STR3## comprisingfermentation of compound (III) ##STR4## with the microorganismActinoplanacete sp. Compounds (I) and (II) and their precursor (III) areAngiotensin II (A II) receptor antagonists useful in the treatment ofhuman hypertensive diseases.

A II is an octapeptide hormone produced mainly in the blood during thecleavage of Angiotensin I by angiotensin converting enzyme (ACE). ACE islocalized on the endothelium of blood vessels of lung, kidney and manyother organs. A II is the end product of the renin-angiotensin system(RAS), a system that plays a central role in the regulation of normalblood pressure and seems to be critically involved in hypertensiondevelopment and maintenance, as well as congestive heart failure. A IIis a powerful arterial vasconstrictor that exerts its action byinteracting with specific receptors present on cell membranes. A IIreceptor antagonism is one of the possible modes of controlling the RAS.

2. Brief Description of Disclosures in the Art

Copending U.S. patent application Ser. No. 370,481, filed Jun. 23, 1989,discloses an hydroxylated analog of simvastatin, obtained from thebioconversion of simvastatin in the presence of Actinoplanacete sp.under controlled fermentation conditions.

Copending U.S. application Ser. No. 516,286, filed May 4, 1990,discloses the substrate compound used in this invention and designatedherein as compound (III), and also generically covers the 5-HOCH₂ -- and7-HOCH₂ -- oxidation products of this invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an ¹ H nuclear magnetic resonance (NMR) spectrum taken at 400MHz of compound (I) in CD₃ OD.

FIG. 2 is an ¹ H NMR spectrum taken at 400 MHz of compound (II) in CD₃OD.

FIG. 3 is an ¹ H NMR spectrum taken at 400 MHz of compound (III) in CD₃OD.

FIG. 4 is a ¹³ C NMR spectrum of compound (II).

SUMMARY OF THE INVENTION

Two new antihypertensive oxidation products, (I) and (II), are obtainedby the fermentation of the microorganism Actinoplanacete sp. (MA 6559),ATCC No. 53771, in the presence of substrate compound (III). Thebiotransformations are accomplished under submerged aerobic conditionsin an aqueous carbohydrate medium containing a nitrogen nutrient at a pHof about 7 for a sufficient time to produce compounds (I) and (II).

Both resultant oxidation products exhibit A II antagonist activity,i.e., both have an inhibitory concentration (IC₅₀) of less than 50 μm inan assay that measures the concentration of potential A II antagonistneeded to achieve 50% displacement of the total ¹²⁵ I-Sar¹ Ile⁸-angiotensin II specifically bound to a membrane preparation derivedfrom rabbit aortae, and therefore both are antihypertensive agents. Thisassay is also referred to herein as the "IC₅₀ " assay.

Also provided by this invention is a pharmaceutical compositioncontaining a therapeutically effective amount of either the 5-HOCH₂ --oxidation product or the 7-HOCH₂ -- oxidation product in combinationwith a pharmaceutically acceptable non-toxic carrier or excipient.

In addition, there is provided a method of treating a human host tocontrol hypertensive disorders or for treating congestive heart failurecomprising administering to said host a therapeutically effective amountof the 5-HOCH₂ -- oxidation product or the 7-HOCH₂ -- oxidation product.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

The products of this invention are compounds (I) and (II): ##STR5##

The novel process of this invention comprises fermentation of themicroorganism Actinoplanacete sp. in the presence of substrate compound(III) ##STR6## in a nutrient medium, and isolation of the resultingbiotransformation products, compounds (I) and (II), in a conventionalmanner. A biologically pure sample of Actinoplanacete sp. is currentlyavailable in the permanent culture collection of the American TypeCulture Collection, 12301 Parklawn Drive in Rockville, Md., from whichit is available under the Accession Number ATCC 53771 as of May 26,1988.

On the basis of the taxonomic analysis performed thus far, themicroorganism Actinoplanacete sp. has tentatively been assigned in theorder Actinomycetales, in the family Actinoplanacea, and in the genusStreptosporangium, and its characteristics are described below:Microscopic observations--Culture grows as branched filaments ranging ofapproximately 6 microns diameter. Spherical to ovoid sporangia aredetected on glycerolasparagine agar, oatmeal agar, yeast-malt extractagar and inorganic salts-starch agar. Sporangia range in size from2.5-44 microns in diameter.

Oat Meal Agar

Vegetative Growth: Reverse is hyaline

Aerial Mass: Moderate, off white, powdery

Soluble Pigment: None

Glycerol-Asparagine

Vegetative Growth: Obverse is mahogany

Aerial Mycelium: Off white and cottony at periphery turning to dustyrose and powdery at colony center

Soluble Pigment: Very light brown

Inorganic Salts-Starch Agar

Vegetative Growth: Mahogany

Aerial Mycelium: Off white and cottony at periphery turning to dustyrose and powdery at colony center

Soluble Pigment: Areas of browning around the periphery of growth withslight clearing of starch

Yeast Extract-Malt Extract Agar

Vegetative Growth: Mahogany to brown black

Aerial Mass: Isolated areas of white, cottony growth against a powderydusty rose colored mycelial matte

Soluble Pigment: Yellow-brown

Egg Albumin Agar

Vegetative Growth: Pale yellow, flat

Aerial Mass: Sparse, white and cottony limited to periphery of growth

Soluble Pigment: None

Nutrient Tyrosine Agar

Vegetative Growth: Transparent to pale yellow

Aerial Mass: None

Soluble Pigment: None

Decomposition of tyrosine: Negative

Skim Milk Agar

Vegetative Growth: Leathery and yellow

Aerial Mass: Sparse, off white and powdery

Soluble Pigment: None

Hydrolysis of casein: Positive

Tomato Paste Oatmeal Agar

Vegetative Growth: Orange-yellow, rugose

Aerial Mass: Powdery, varying in color from off white to purple-brown

Gelatin Stabs

Vegetative Growth: Orange yellow

Aerial Mass: None

Soluble Pigment: None

Liquification of gelatin: Positive

Peptone-Iron-Yeast Extract Agar Slants

Vegetative Growth: Colorless, leathery

Aerial Mass: Moderate, off white, powdery

Soluble Pigment: None

Melanin: Negative

H₂ S: Negative

Tryptone Yeast Extract Broth

Soluble Pigment: None

    ______________________________________                                        Carbohydrate utilization pattern                                              ______________________________________                                        d-glucose                                                                             ++      d-maltose +    sucrose   +/-                                  d-arabinose                                                                           ++      d-mannitol                                                                              ++   d-xylose  ++                                   l-arabinose                                                                           ++      d-mannose ++   l-xylose  -                                    d-fructose                                                                            ++      l-mannose -    alpha d-lactose                                                                         ++                                   l-glucose                                                                             +/-     d-raffinose                                                                             ++   beta d-lactose                                                                          ++                                   inositol                                                                              +       l-rhamnose                                                                              -                                                   ______________________________________                                    

Carbon source utilization studies were carried out using Pridham andGottlieb basal medium supplemented with 1% carbon source. Scoring wasgraded according to the methods described in "Methods forCharacterization of Streptomyces species", IJSB 16: pps 313-340.

In general, the 5-HOCH₂ -- and the 7-HOCH₂ -- oxdation products can beproduced by culturing (fermenting) the above-described microorganism inthe presence of an appropriate concentration of substrate compound (III)in an aqueous nutrient medium containing sources of assimilable carbonand nitrogen, preferably under submerged aerobic conditions (e.g.shaking culture, submerged culture, etc.). An appropriate concentrationof the parent compound in the aqueous medium ranges from 0.05 mg/ml to0.2 mg/ml, preferably 0.1 mg/ml; less than 0.05 mg/ml is inefficient andgreater than 0.2 mg/ml can inhibit the culture. The aqueous medium isincubated at a temperature between 26° C. and 29° C., preferably 27° C.;culture growth will be inhibited below this temperature range andculture death will occur above this temperature range. The aqueousmedium is incubated for a period of time necessary to complete theoxidative biotransformations as monitored by HPLC, usually for a periodof about 24 hours, on a rotary shaker operating at about 220 rpm. with athrow of about 2 in. The aqueous medium is maintained at a pH between 6and 8, preferably about 7, at the initiation and termination (harvest)of the fermentation process. A higher or lower pH will cause the cultureto die. The desired pH may be maintained by the use of a buffer such asmorpholinoethanesulfonic acid (MES), morpholinopropanesulfonic acid(MOPS), and the like, or by choice of nutrient materials whichinherently possess buffering properties, such as production mediadescribed herein below.

The preferred sources of carbon in the nutrient medium are certaincarbohydrates such as glucose, xylose, galactose, glycerin, starch,dextrin, and the like. Other sources which may be included are maltose,rhamnose, raffinose, arabinose, mannose, salicin, sodium succinate, andthe like.

The preferred sources of nitrogen are yeast extract, meat extract,peptone, gluten meal, cottonseed meal, soybean meal and other vegetablemeals (partially or totally defatted), casein hydrolysates, soybeanhydrolysates and yeast hydrolysates, corn steep liquor, dried yeast,wheat germ, feather meal, peanut powder, distiller's solubles, etc., aswell as inorganic and organic nitrogen compounds such as ammonium salts(e.g. ammonium nitrate, ammonium sulfate, ammonium phosphate, etc.),urea, amino acids, and the like.

The carbon and nitrogen sources, though advantageously employed incombination, need not be used in their pure form because less purematerials which contain traces of growth factors and considerablequantities of mineral nutrients are also suitable for use. When desired,there may be added to the medium mineral salts such as sodium or calciumcarbonate, sodium or potassium phosphate, sodium or potassium chloride,sodium or potassium iodide, magnesium salts, copper salts, cobalt salts,and the like. If necessary, especially when the culture medium foamsseriously, a defoaming agent, such as liquid paraffin, fatty oil, plantoil, mineral oil or silicone may be added.

Submerged aerobic cultural conditions are preferred for the productionof the 5-HOCH₂ -- and 7-HOCH₂ -- oxidation products in massive amounts.For the production in small amounts, a shaking or surface culture in aflask or bottle is employed. Furthermore, when the growth is carried outin large tanks, it is preferable to use the vegetative form of theorganism for inoculation in the production tanks in order to avoidgrowth lag in the process of production of the 5-HOCH₂ -- and 7-HOCH₂ --oxidation products. Accordingly, it is desirable first to produce avegetative inoculum of the organism by inoculating a relatively smallquantity of culture medium with spores or mycelia of the organismproduced in a "slant" and culturing said inoculated medium, also calledthe "seed medium", and then to transfer the cultured vegetative inoculumaseptically to large tanks. The fermentation medium, in which theinoculum is produced, is substantially the same as or different from themedium utilized for the production of the 5-HOCH₂ -- and 7-HOCH₂ --oxidation products and is generally autoclaved to sterilize the mediumprior to inoculation. The fermentation medium is generally adjusted to apH between 6 and 8, preferably about 7, prior to the autoclaving step bysuitable addition of an acid or base, preferably in the form of abuffering solution. Temperature of the seed medium is maintained between26° C. and 29° C., preferably 27° C.; culture growth will be inhibitedbelow this range and culture death will occur above this range.Incubation of the seed medium is usually conducted for a period of about10 to 30 hours, preferably 24 hours, on a rotary shaker operating at 220rpm; the length of incubation time may be varied according tofermentation conditions and scales. Agitation and aeration of theculture mixture may be accomplished in a variety of ways. Agitation maybe provided by a propeller or similar mechanical agitation equipment, byrevolving or shaking the fermentor, by various pumping equipment or bythe passage of sterile air through the medium. Aeration may be effectedby passing sterile air through the fermentation mixture.

Preferred culturing/production media for carrying out the fermentationinclude the following media:

    ______________________________________                                                          g/l                                                         ______________________________________                                        Seed Medium A                                                                 Dextrose            1.0                                                       Dextrin             10.0                                                      Beef Extract        3.0                                                       Ardamine pH         5.0                                                       NZ Amine Type E     5.0                                                       MgSO.sub.4.7H.sub.2 O                                                                             0.05                                                      K.sub.2 HPO.sub.4   0.37                                                      Adjust pH to 7.1                                                              Add CaCO.sub.3 0.5 g/l                                                        Transformation Medium B                                                       Glucose             10                                                        Hycase SF           2                                                         Beef Extract        1                                                         Corn Steep Liquor   3                                                         Adjust pH to 7.0                                                              ______________________________________                                    

The produced 5-HOCH₂ -- and 7-HOCH₂ -- oxidation products can berecovered from the culture medium by conventional means which arecommonly used for the recovery of other known biologically activesubstances. The 5-HOCH₂ -- and 7-HOCH₂ -- oxidation products are foundin the cultured mycelium and filtrate, which are obtained by filteringor centrifuging the cultured broth, and accordingly can be isolated andpurified from the mycelium and the filtrate by a conventional methodsuch as concentration under reduced pressure, lyophilization, extractionwith a conventional solvent, such as methylene chloride and the like, pHadjustment, treatment with a conventional resin (e.g. anion or cationexchange resin, non-ionic adsorption resin, etc.), treatment with aconventional adsorbent (e.g. activated charcoal, silicic acid, silicagel, cellulose, alumina, etc.), crystallization, recrystallization, andthe like. A preferred recovery method is solvent extraction,particularly using methylene chloride. A preferred purification methodinvolves the use of chromatography, especially HPLC, using a silica gelcolumn and an eluant mixture composed of water and an organic solventsuch as methanol, acetonitrile and the like, and a small amount of acidsuch as acetic acid, trifluoracetic acid, phosphoric acid and the like.A preferred eluant is composed of water, acetonitrile, and 0.1%trifluoroacetic acid and is run through the column in a linear gradient.

The compounds of this invention form salts with various inorganic andorganic acids and bases which are also within the scope of theinvention. Such salts include ammonium salts, alkali metal salts likesodium and potassium salts, alkaline earth metal salts like the calciumand magnesium salts, salts with organic bases; e.g., dicyclohexylaminesalts, N-methyl-D-glucamine, salts with amino acids like arginine,lysine and the like. Also, salts with organic and inorganic acids may beprepared; e.g., HCl, HBr, H₂ SO₄, H₃ PO₄, methane-sulfonic,toluene-sulfonic, maleic, fumaric, camphorsulphonic. The non-toxic,physiologically acceptable salts are preferred.

The salts can be formed by conventional means such as by reacting thefree acid or free base forms of the product with one or more equivalentsof the appropriate acid or base in a solvent or medium in which the saltis insoluble, or in a solvent such as water which is then removed invacuo or by freeze-drying, or by exchanging the cations of an existingsalt for another cation on a suitable ion exchange resin.

Suitable formulations of the material may also include conventionalpharmaceutically acceptable biolabile esters of compounds (I) and (II)formed via the hydroxy groups on the molecule, such as the acetate.

The 5-HOCH₂ -- and 7-HOCH₂ -- oxidation products of this invention bothexhibit A II antagonist activity by the IC₅₀ assay, and therefore bothare useful in treating hypertension. They are also of value in themanagement of acute and chronic congestive heart failure, in thetreatment of secondary hyperaldosteronism, primary and secondarypulmonary hyperaldosteronism, primary and secondary pulmonaryhypertension, renal failure, renal vascular hypertension, ocularhypertension and impaired retinal blood flow, and in the management ofvascular disorders such as migraine or Raynaud's disease. Theapplication of the compounds of this invention for these and similardisorders will be apparent to those skilled in the art.

In the management of hypertension and the clinical conditions notedabove, the compounds of this invention may be utilized in compositionssuch as tablets, capsules or elixirs for oral administration,suppositories for rectal administration, sterile solutions orsuspensions for parenteral or intramuscular administration, and thelike. The compounds of this invention can be administered to patients(animals and human) in need of such treatment in dosages that willprovide optimal pharmaceutical efficacy. Although the dose will varyfrom patient to patient depending upon the nature and severity ofdisease, the patient's weight, special diets then being followed by apatient, concurrent medication, and other factors which those skilled inthe art will recognize, the dosage range will generally be about 1 to1000 mg. per patient per day which can be administered in single ormultiple doses. Perferably, the dosage range will be about 2.5 to 250mg. per patient per day; more preferably about 2.5 to 75 mg. per patientper day.

The compounds of this invention can also be administered in combinationwith other antihypertensives such as α-methyldopa, and/or diuretics suchas hydrochlorothiazide, and/or angiotensin converting enzyme inhibitorssuch as enalapril, and/or calcium channel blockers such as nifedipine.Typically, the individual daily dosages for these combinations can rangefrom about one-fifth of the minimally recommended clinical dosages tothe maximum recommended levels for the entities when they are givensingly. These dose ranges can be adjusted on a unit basis as necessaryto permit divided daily dosage and, and as noted above, the dose willvary depending on the nature and severity of the disease, weight of thepatient, special diets and other factors. Typically, these combinationscan be formulated into pharmaceutical compositions as discussed below.

About 1 to 100 mg. of either compound (I) or (II) or a mixture ofcompounds (I) and (II) or a physiologically acceptable salt thereof iscompounded with a physiologically acceptable vehicle, carrier,excipient, binder, preservative, stabilizer, flavor, etc., in a unitdosage form as called for by accepted pharmaceutical practice. Theamount of active substance in these compositions or preparations is suchthat a suitable dosage in the range indicated is obtained.

Illustrative of the adjuvants which can be incorporated in tablets,capsules and the like are the following: a binder such as gumtragacanth, acacia, corn starch or gelatin; an excipient such asmicrocrystalline cellulose; a disintegrating agent such as corn starch,pregelatinized starch, alginic acid and the like; a lubricant such asmagnesium stearate; a sweetening agent such as sucrose, lactose orsaccharin; a flavoring agent such as peppermint, oil of wintergreen orcherry. When the dosage unitform is a capsule, it may contain, inaddition to materials of the above type, a liquid carrier such as fattyoil. Various other materials may be present as coatings or to otherwisemodify the physical form of the dosage unit. For instance, tablets maybe coated with shellac, sugar or both. A syrup or elixir may contain theactive compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and a flavoring such as cherry ororange flavor.

Sterile compositions for injection can be formulated according toconventional pharmaceutical practice by dissolving or suspending theactive substance in a vehicle such as water for injection, a naturallyoccuring vegetable oil like sesame oil, coconut oil, peanut oil,cottonseed oil, etc., or a synthetic fatty vehicle like ethyl oleate orthe like. Buffers, preservatives, antioxidants and the like can beincorporated as required.

The compounds of this invention are also useful to treat elevatedintraocular pressure and can be administered to patients in need of suchtreatment with typical pharmaceutical formulations such as tablets,capsules, injectables, as well as topical ocular formulations in theform of solutions, ointments, inserts, gels, and the like.Pharmaceutical formulations prepared to treat intraocular pressure wouldtypically contain about 0.9% to 15% by weight, preferably 0.5% to 2% byweight, of a compound of this invention.

Receptor Binding Assay Using Rabbit Aortae Membrane Preparation

Three frozen rabbit aortae (obtained from Pel-Freeze Biologicals) weresuspended in 5 mM Tris-0.25M Sucrose, pH 7.4 buffer (50 ml) homogenized,and then centifuged. The mixture was filtered through a cheesecloth andthe supernatant was centrifuged for 30 minutes at 20,000 rpm at 4° C.The pellet thus obtained was resuspended in 30 ml of 50 mM Tris-5 mMMgCl₂ buffer containing 0.2% Bovine Serum Albumin and 0.2 mg/mlBacitration and the suspension was used for 100 assay tubes. Samplestested for screening were done in duplicate. To the membrane preparation(0.25 ml) there was added ¹²⁵ I-Sar¹ Ile⁸ -angiotensin II [obtained fromNew England Nuclear] (10 ul; 20,000 cpm) with or without the test sampleand the mixture was incubated at 37° C. for 90 minutes. The mixture wasthen diluted with ice-cold 50 mM Tris---0.9% NaCl, pH 7.4 (4 ml) andfiltered through a glass fiber filter (GF/B Whatman 2.4" diameter). Thefilter was soaked in scintillation cocktail (10 ml) and counted forradioactivity using Packard 2260 Tricarb liquid scintillation counter.The inhibitory concentration (IC₅₀) of potential AII antagonist whichgives 50% displacement of the total specifically bound ¹²⁵ I-Sar¹ Ile⁸-angiotensin II was presented as a measure of the efficacy of suchcompounds as AII antagonists.

Using the methodology described above, both of the compounds of theinvention were evaluated and both were found to exhibit an activity ofat least IC₅₀ <50 μm thereby demonstrating and confirming the utility ofthe compounds of the invention as effective AII antagonists.

Preparation of Substrate Compound (III):5,7-Dimethyl-2-ethyl-3-(2'-(tetrazol-5-yl)biphen-4-yl)methyl-3H-imidazo[4,5-b]pyridineI. Preparation ofN-Triphenylmethyl-5-(4'-bromomethyl-biphen-2-yl)tetrazole

Step 1: 2-cyano-4'-methylbiphenyl

To a solution of p-bromotoluene (30 g) in dry ether (150 ml) at -78° C.,a solution of t-BuLi in pentane (1.7M) (210 ml) was added slowly over aperiod of 1 hour and 30 minutes, using a dropping funnel. The bath wasthen removed and the mixture was stirred at room temperature for anadditional 2 hours. The content of the flask was then added slowly(using a cannula) at room temperature to a premixed solution of ZnCl₂ inether (1M) (180 mL) and dry THF (360 mL). The mixture was stirred for 2hours at that temperature and the slurry was added (using a cannula) toa solution of 2-bromobenzonitrile (21.3 g) and NiCl₂ (Ph₃ P)₂ (2.1 g) indry THF (300 ml). The mixture, after stirring at room temperatureovernight (18 hours), was poured slowly with stirring into ice cold 0.5NHCl (1500 ml). The organic layer was separated, and the aqueous phasewas extracted with ether (3×300 ml). The combined organic layer waswashed with water, brine and then dried over MgSO₄. Removal of thesolvent gave the crude product as a semisolid mass (34 g). The materialwas purified on a silica gel flash column using ethylacetate/hexane(1:12) to give the desired nitrile as a low melting solid (28 g, 88%).NMR (CDCl₃) δ 2.42 (s, 3H), 7.2-7.8 (m, 8H); FAB-MS: m/e 194 (M+H).

Step 2: Trimethylstannyl azide

To a concentrated solution of NaN₃ (40 g) in water (100 ml), a solutionof trimethyltin chloride (20 g) in dioxane (10 ml) was added in threeportions under vigorous stirring. An instantaneous precipitate formationwas observed. The mixture, after stirring overnight at room temperature,was filtered. The residue was washed with water, and dried under suctionand then in vacuo over P₂ O₅. Yield 18.7 g (81%), mp 132°-136° C.

Step 3: N-Triphenylmethyl-5-(4'-bromomethyl-biphen-2-yl)tetrazole

The titled compound was prepared starting from 2-cyano-4'-methylbiphenyl(Step 1) as described in European Patent Application EP 0,291,969.

II. Preparation of 5,7-Dimethyl-2-ethylimidazo[4,5-b]pyridine

Step 1: 2-Amino-4,6-dimethyl-3-nitropyridine

2-Amino-4,6-dimethylpyridine (10.0 g, 81.8 mmol) was added portion-wiseto 65 mL of H₂ SO₄ (conc. d=1.84) which was stirred (mechanical) at 0°C. After complete addition, the mixture was warmed to room temperatureuntil the mixture became homogeneous. The solution was then cooled to-10° C. and a pre-cooled (0° C.) mixture of concentrated HNO₃ (11.5 mL,d=1.40) and H₂ SO₄ (8.2 mL, d=1.84) was added at such a rate as not toraise the internal reaction temperature above -9° C. Ten minutes afterthe addition was complete this cooled (-10° C.) mixture was poured onto400 g of crushed ice. The resulting slurry was neutralized by theaddition of conc NH₄ OH (to pH 5.5) while cooling (ice bath). The solidwas isolated by filtration, and dried at room temperature to give 13.3 gof 2-nitramino-4,6-dimethylpyridine as a white solid.

To 75 mL of stirred conc H₂ SO₄ cooled to -5° C. (ice-salt bath) wasadded 4,6-2-nitraminopyridine (13.2 g, 79 mmol) portion-wise at such arate as to maintain the internal temperature below -3° C. The mixturewas warmed to 0° C. until homogeneous (30 minutes) at which time tlc(SiO₂, 1:1 EtOAc/hexanes on a NH₄ OH neutralized aliquot) indicated thatthe rearrangement was complete. The mixture was poured onto 400 g ofcrushed ice and the pH was adjusted to 5.5 by the addition ofconcentrated NH₄ OH. The resulting yellow slurry was cooled to 0° C.,filtered, washed with cold water (50 mL), and dried at room temperatureto give 10.32 g of a mixture of the title compound and the 5-nitroisomer in a 55:45 ratio (determined by ¹ H NMR). This mixture was useddirectly in Step 2.

Step 2: 5,7-Dimethyl-2-ethylimidazo[4,5-b]pyridine

To a mixture of 8.44 g of a 55:45 mixture of2-Amino-3-nitro-4,6-dimethylpyridine and2-Amino-4,6-dimethyl-5-nitropyridine in MeOH (1.2 L) was added 10% Pd/C(2.4 g). The reaction vessel was evacuated then purged with H₂ at 1 atm.and stirred vigorously for 18 hours. Filtration (celite), andconcentration gave 6.65 g of a mixture of2,3-diamino-4,6-dimethylpyridine and 2,5-diamino-4,6-dimethylpyridine asa dark solid. To 5.40 g (39.4 mmol) of this mixture was added propionicacid (8.80 mL, 118 mmol) followed by polyphosphoric acid (100 mL). Thisstirred mixture was heated to 90° C. for 3 hours then to 100° C. for 1hour. After the reaction was complete, the warm mixture was poured onto300 g of ice and the mixture was made basic with NH₄ OH. The mixture wasextracted (4×50 mL CH₂ Cl₂), dried (K₂ CO₃) and concentrated to give amixture of the title compound and4,6-dimethyl-2,5-bis(propionamido)pyridine. Purification (SiO₂, 5%MeOH/EtOAc) gave 1.66 g of the title compound as the slower elutingcomponent. ¹ H NMR (CD₃ OD, 300MHz) δ 6.95 (s, 1H), 2.92 (q, 2H, J=7.8Hz), 2.54 (apparent s, 6H), 1.40 (t, 3H, J=7.8 Hz)

III.5,7-Dimethyl-2-ethyl-3-(2'-(tetrazol-5-yl)biphen-4-yl)methyl-3H-imidazo[4,5-b]-pyridine

Part A

To a stirred suspension of NaH (17.2 mmol of an 80% dispersion) in drydimethylformamide (30 mL) at rt was added5,7-dimethyl-2-ethylimidazo[4,5-b]pyridine (1.51 g, 8.62 mmol) in oneportion. After 20 minutes, the mixture was cooled to 0° C. andN-triphenylmethyl-5-(4'-bromomethylbiphenyl-2-yl)tetrazole (5.29 g, 9.48mmol) was added in one portion. The resulting mixture was warmed to rtand stirred for 15 hours. The excess NaH was quenched with water and thebulk of the DMF was removed in vacuo at 40°-50° C. The residue wasextracted with EtOAc from brine, dried (K₂ CO₃), and concentrated.Purification by flash chromatography (SiO₂, solvent gradient: 80%EtOAc/hexanes, 100% EtOAc) gave 4.25 g of5,7-dimethyl-2-ethyl-3-(2'-(N-triphenylmethyltetrazol-5-yl)biphen-4-yl)methyl-3H-imidazo[4,5-b]pyridine:¹ H NMR (300 MHz, CDCl₃) δ 7.86 (dd, 1H, J=7, 2 Hz), 7.50-7.41 (m, 2H),7.36-7.21 (m, 10H), 7.05 (d, 2H, J=4.5 Hz), 6.95-6.89 (m, 7H), 6.86 (d,2H, J=4.5 Hz), 5.35 (s, 2H), 2.67 (q, 2H, J=7.5 Hz), 2.65 (s, 3H), 2.58(s, 3H), 1.25 (t, 3H, J=7.5 Hz).

Part B

To a stirred solution of the trityl-protected tetrazole (4.13 g, 6.33mmol) in CH₂ Cl₂ (40 mL) at room temperature was added 85% formic acid(60 mL). After 45 minutes, the mixture was concentrated and the residuewas purified by chromatography (SiO₂, 85:13.5:1.5 CHCl₃ --MeOH--NH₄ OH)followed by crystallization from 30 mL of MeOH to give 2.18 g (84%)solid: mp 156°-158° C. ¹ H NMR (300 MHz, CD₃ OD) δ 7.68-7.61 (m, 2H),7.57-7.50 (m, 2H), 7.07 (apparent singlet, 4H), 7.04 (s, 1H), 5.55 (s,2H), 2.85 (q, 2H, J=7.5 Hz), 2.61 (s, 3H), 2.58 (s, 3H), 1.25 (t, 3H,J=7.5 Hz).

Anal. Calcd for C₂₄ H₂₃ N₇.0.25 H₂ O: C, 69.63; H, 5.72; N, 23.68.Found: C, 69.91; H, 5.73; N, 23.60.

The following examples are given for the purpose of illustrating thepresent invention and should not be construed as being limitations onthe scope or spirit of the instant invention.

EXAMPLE 1 Microorganism and Culture Conditions

A frozen vial (2.0 ml) of culture (MA 6659) ATCC No. 53771 was used toinoculate a 250 ml baffled shake flask containing 50 ml of an autoclaved(sterilized) seed medium consisting of (in units of grams/liter) dextrin10.0, dextrose 1.0, beef extract 3.0, ardamine PH (Yeast Products, Inc.)5.0, N-Z Amine type E 5.0, MgSO₄.7H₂ O 0.05, K₂ HPO₄ 0.3, and CaCO₃ 0.5.The pH of the seed medium was adjusted to 7.1 before autoclaving. Theseed was incubated in the seed medium at 27° C. for 24 hours on a rotaryshaker operating at 220 rpm. A 2.5 ml aliquot of the resulting seedmedium was used to inoculate a 250 ml non-baffled shake flask containing50 ml of the following previously autoclaved (sterilized) transformationmedium B.* Substrate compound (III) was added as an aqueous solutionwith pH of 8 to achieve a final concentration of 0.1 mg/ml. The shakeflask contents were subsequently incubated for 24 hours at 27° C. on arotary shaker operating at 220 rpm. This procedure was followed threetimes and the three resultant broths were combined for isolation andpurification.

Isolation and Purification Procedure for the Broth

The whole broth (150 ml) of transformation media B was acidified to pH3.5 and then extracted three times with methylene chloride (3×100 ml).Methylene chloride extracts were combined, dried over sodium sulfate,and concentrated under vacuum to an oily residue. The residue wasdissolved in methanol and subjected to high performance liquidchromatography (HPLC) purification.

HPLC was carried out on Whatman Partisil 10ODS-3, 9.4 mm×25 cm column atroom temperature and monitored at 255 nm and 275 nm. The column wasdeveloped at 3 ml./min with linear gradient from 0.1% aqueous TFA-CH₃CN, 85:15 to 0.1% aqueous TFA-CH₃ CN, 15:85 in 45 minutes. The compoundswere collected during repeated injections of the above describedextract. Both compounds were visible at 255 nm and 275 nm. The fractionsat retention time 17.5 and 19.8 minutes were pooled respectively, andevaporated to remove solvents to yield 6.0 mg. of compound (I)characterized as the 5-HOCH₂ -- oxidation product, and 3.0 mg ofcompound (II) characterized as the 7-HOCH₂ -- oxidation product,respectively.

Characterization

Compound (I) of this invention was characterized via NMR spectrometryyielding the proton NMR spectrum of FIG. 1, which also contains theassigned molecular structure.

Compound (II) of this invention was characterized via NMR spectrometryyielding the proton NMR spectrum of FIG. 2 which includes the assignedmolecular structure and the ¹³ C NMR of FIG. 4.

What is claimed:
 1. A process for the preparation of a compoundrepresented by formula (I) or (II) ##STR7## comprising the steps ofculturing a microorganism Actinoplanacete sp. (MA 6559) (ATCC 53771) ina nutrient medium containing assimilable sources of nitrogen and carbonand substrate compound (III) ##STR8## under aerobic conditions until asubstantial amount of the compounds are produced and isolating thecompounds so produced.
 2. The process of claim 1 wherein the temperatureis 26°-29° C.
 3. The process of claim 2 wherein the temperature is 27°C.